Detection of bacteria using bacteriophage

ABSTRACT

A method of detecting a species, strain or type of bacteria includes mixing a labeled bacteriophage including a label that is detectible via a detection system with a bacterial culture including the species, strain or type of bacteria to which the labeled bacteriophage selectively binds and using the detection system to detect the labeled bacteriophage bound to the species, strain or type of bacteria.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Patent ApplicationSer. No. 62/199,472, filed Jul. 31, 2015, the disclosure of which isincorporated herein by reference.

BACKGROUND

The following information is provided to assist the reader inunderstanding technologies disclosed below and the environment in whichsuch technologies may typically be used. The terms used herein are notintended to be limited to any particular narrow interpretation unlessclearly stated otherwise in this document. References set forth hereinmay facilitate understanding of the technologies or the backgroundthereof. The disclosure of all references cited herein are incorporatedby reference.

Current methods for isolating bacteria from, for example, body fluids(such as blood) require 48 hours or longer in a hospital, laboratory orother setting to determine the exact species, strain or type of bacteriapresent. Moreover, those methods are unable to quantify accurately anydetected bacteria and are limited to those that readily grow on an agarplate. A quicker, quantitative assay or test would be highly beneficial.

SUMMARY

In one aspect, a method of detecting or determining a species, strain ortype of bacteria includes mixing a labeled bacteriophage including alabel that is detectible via a detection system with a bacterial cultureincluding the species, strain or type of bacteria to which the labeledbacteriophage selectively binds and using the detection system to detectthe labeled bacteriophage bound to the species, strain or type ofbacteria. The method may, for example, further include removing unboundlabeled bacteriophage after mixing the labeled bacteriophage with thebacterial culture. In a number of embodiments, the detection systemcomprises a photoacoustic cell. The detection system may, for example,include a photoacoustic flowmetry system. The species, strain or type ofbacteria may be identified and quantified. In a number of embodiments,the level of energy used in a photoacoustic system (or a systemincluding a photoacoustic cell) is sufficiently low to reduce, minimizeor eliminate detection of unbound labeled bacteriophage. In that regard,by binding to a target bacterium, the bacteriophage becomespatially-sequestered, thus enhancing the signal their tag produces.

In a number of embodiments, a plurality of labeled bacteriophages may bemixed with the sample. Each of the plurality of bacteriophagesselectively binds with a different species, strain or type of bacteria.Each of the plurality of bacteriophages includes a different label thatis separately detectable via the detection system. The detection systemis used to determine the presence of the labeled bacteriophage bound toat least one of the different species, strains or types of bacteria. Thebacteriophages may, for example, be labeled with different labels thatare detectible at different wavelengths of energy.

In another aspect, a system for determining if a sample includes atleast one species, strain or type of bacteria, includes a plurality ofbacteriophages. Each of the plurality of bacteriophages selectivelybinding with a different species, strain or type of bacteria. Each ofthe plurality of bacteriophages including a different label that isseparately detectable via a detection system.

In another aspect, a system for detecting a species, strain or type ofbacteria includes a labeled bacteriophage, which binds selectively tothe species, strain or type of bacteria and includes a label attachedthereto, and a system adapted to detect the labeled bacteriophage boundto the species, strain or type of bacteria. The detection system may,for example, include a photoacoustic cell. In a number of embodiments,the detection system includes a photoacoustic flowmetry system. Asdescribed above, the species, strain or type of bacteria may beidentified and quantified.

In a further aspect, a bacteriophage includes a label attached thereto,wherein the label is selected to be detectible via a detection system.The label may, for example, be detectible using a photoacoustic cell. Ina number of embodiments, the label is detectible using a photoacousticflowmetry system.

The present devices, systems, methods and compositions, along with theattributes and attendant advantages thereof, will best be appreciatedand understood in view of the following detailed description taken inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a representative scheme for labeling or tagging abacteriophage with a label including an isothiocyanate moiety(fluorescein isothiocyanate).

FIG. 2 illustrates an embodiment of a photoacoustic flowmetry system.

FIG. 3 illustrates a study of the use of labeled or tagged bacteriophageto detect a specific bacteria using a photoacoustic flowmetry systemshowing that untagged bacteria do not provide a photoacoustic signalwhereas tagged bacteria (via the tagged bacteriophage) provide aphotoacoustic signal.

FIG. 4 illustrates a representation of isolation of target bacterialcells using two phase flow.

FIG. 5 illustrates another representation of isolation of targetbacterial cells using two phase flow.

FIG. 6A illustrates a waveform resulting from laser irradiation ofbacteriophage at about 2.12 millijoules (mJ) per pulse.

FIG. 6B illustrates a waveform resulting from laser irradiation ofbacteriophage at about 3.96 mJ per pulse.

DETAILED DESCRIPTION

It will be readily understood that the components of the embodiments, asgenerally described and illustrated in the figures herein, may bearranged and designed in a wide variety of different configurations inaddition to the described representative embodiments. Thus, thefollowing more detailed description of the representative embodiments,as illustrated in the figures, is not intended to limit the scope of theembodiments, as claimed, but is merely illustrative of representativeembodiments.

Reference throughout this specification to “one embodiment” or “anembodiment” (or the like) means that a particular feature, structure, orcharacteristic described in connection with the embodiment is includedin at least one embodiment. Thus, the appearance of the phrases “in oneembodiment” or “in an embodiment” or the like in various placesthroughout this specification are not necessarily all referring to thesame embodiment.

Furthermore, described features, structures, or characteristics may becombined in any suitable manner in one or more embodiments. In thefollowing description, numerous specific details are provided to give athorough understanding of embodiments. One skilled in the relevant artwill recognize, however, that the various embodiments can be practicedwithout one or more of the specific details, or with other methods,components, materials, et cetera. In other instances, well knownstructures, materials, or operations are not shown or described indetail to avoid obfuscation.

As used herein and in the appended claims, the singular forms “a,” “an”,and “the” include plural references unless the context clearly dictatesotherwise. Thus, for example, reference to “a bacteriophage” includes aplurality of such bacteriophages and equivalents thereof known to thoseskilled in the art, and so forth, and reference to “the bacteriophage”is a reference to one or more such bacteriophages and equivalentsthereof known to those skilled in the art, and so forth. Recitation ofranges of values herein are merely intended to serve as a shorthandmethod of referring individually to each separate value falling withinthe range. Unless otherwise indicated herein, and each separate value,as well as intermediate ranges, are incorporated into the specificationas if individually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contraindicated by the text.

Bacteriophages (sometime referred to as phages) are viruses that infectbacteria by discriminately binding to externally presented surfaceantigens of their target bacterial hosts. In a number of embodiments,devices, systems and methods hereof provide rapid bacterial typing assayusing a sensing system (for example, a photoacoustic sensing system) andlabeled bacteriophages. The terms “label” and “tag” are used hereininterchangeably to refer to an entity or moiety that is associable witha bacteriophage and that can be detected via a detection system. In thatregard, bacteriophages or phages are labeled or tagged using adetectible label or tag (for example, a photoacoustic labile label ortag) and then added to, for example, bacterial mixtures or cultures oftarget and non-target bacteria. As used herein, the term “labile” refersto labels or tags which may be easily changed and/or the same label ortag may be used with a variety of bacteriophages. A wash step may, forexample, be used to remove excess and unbound phage after phageabsorption. The labeled or tagged phage/bacterial mixture is thenprocessed through a detection system (such as a photoacoustic cell)where targeted bacterias are detected by the attached labeled or taggedphage.

Photoacoustics is the transduction of photons into mechanical energy bytargeting light absorbing objects with rapid pulsed laser light.Photoacoustic waves may, for example, be generated by thermoelasticexpansion, that is laser induced heating, resulting in volume expansionand contraction.

Studies of a number of embodiments hereof have demonstrated thefeasibility of the present approach with, for example, a spiked culturewith a signal to noise ratio in excess of 5/1. The studies hereofdemonstrate that bacteriophage can be effectively used to determinebacterial contamination in a mixed culture. Moreover, the studies hereofdemonstrate the value of expanding detection capabilities by usingadditional phages with varying host ranges targeting, for example, bothgram negative and gram positive human pathogens. The devices, systemsand methods hereof provide a clinically accessible method to determinebacterial contamination and lower the time required to obtain results(as compared to present detection methodologies) by at least one orderof magnitude (for example, results within 3-4 hours rather than 3-4days). An assay with such capabilities may be very advantageous in, forexample, hospitals and medical laboratories. Incorporating assays hereofmay, for example, allow doctors to treat infections earlier using, forexample, targeted antibiotics. The impact of such advancement indetection techniques would be likely to support future tailored phagetherapy and have a significant and global lifesaving potential.

Host ranges for a variety of bacteriophages have been determinedpreviously. Additionally, bacterial host range has been shown to beprimarily determined by phage binding to bacterial O-antigens and othersurface proteins presented by the bacteria. As set forth above,bacteriophage are viruses that infect bacteria. The host range of abacteriophage is defined by which bacterial genera, species and strainsor types the bacteriophage can infect and lyse. The host range of abacteriophage is one of the defining biological characteristics of aparticular bacterial virus. Productive phage infection requires: 1.successful host attachment, 2. initial penetration of the host outermembrane and/or cell wall, 3. transfer of the phage DNA through theinner membrane leading to synthesis of phage-encoded proteins andnucleic acid, and 4. escape of progeny phage from the dead host cell(lysis). Host attachment is mediated by specialized proteins called tailspikes and tail fibers presented on the posterior tip of tailed phages.These proteins attach to specific structures on the outer surface ofhost bacteria. Lipopolysaccharides (O-antigens), flagellin, teichoicacids, capsular polysaccharides (K-antigens), and specific membraneproteins such as nutrient transporters can be used as attachment sites.The ability of a phage to attach to specific cell surface molecules isconsidered the primary determinant of host range. There are otherphysiological or genetic properties of cells that influence the abilityof certain phage to replicate in them, for example the presence of arestriction enzyme or a CRISPR system, and these can in principleinfluence host range. Bacteriophage host range and bacterial resistanceis, for example, discussed in Hyman P1, Abedon S T, Adv Appl Microbiol.m 2010; 70:217-48. doi: 10.1016/S0065-2164(10)70007-1. Epub 2010 Mar. 6;and Kutter, E. Methods Mol Biol., 2009; 501:141-9. doi:10.1007/978-1-60327-164-6_14.

By using, for example, a photoacoustic cell or other detection system,one may use a bacteriophage's ability to discriminately or selectivelybind to further discriminate the bacteria. In that regard, a labeled ortagged (for example, photoacoustic labeled or tagged) bacteriophageallows one to detect or identify specific bacteria to which the labeledbacteriophage selectively binds. Labels and associated detection systemsother than photoacoustic detections system (such as flow cytometry,using a fluorescent label or tag), or electron microscopy may be used ina number of embodiments hereof. In flow cytometry, a bacteriophage may,for example, have a fluorescent molecule bound to the bacteriophageusing an intermediary binding agent (for example, monoclonal antibody tophage and green fluorescent protein or GFP bound to antibody). Electronmicroscopy may, for example, be used wherein nano-gold particles may beattached to bacteriophage, targeted to bacteria, stained, and countedusing an electron microscope. In general, photoacoustic sensing ordetection systems may provide increased detection rates and specificityover many other detection systems (for example, flow cytometry and orelectron microscopy) at a reduced cost.

In a number of studies hereof, a photoacoustic compatible orphotoacoustic label/tag was first bound to bacteriophage. Aphotoacoustic tag or label as used herein refers to any compound ormoiety that provides a photoacoustic signal. Such compounds can bedetermined in the literature, via experimentation, or via theory. In anumber of embodiments, photoacoustic tags or labels suitable for useherein are readily incorporated into or bound to the bacteriophage anddo not interfere significantly with the binding activity of thebacteriophage. Examples of suitable photoacoustic tags or labelsinclude, but are not limited to, fluorescein isothiocyanate (FITC;available, for example, from Sigma-Aldrich of St. Louis, Mo. USA), Evansblue dye (EB; an azo dye having the formula C₃₄H₂₄N₆Na₄O₁₄S₄; available,for example, from Sigma-Aldrich), IR775S, Blue (a cyanine dyederivative) and the protein dye Direct Red 81 (C₂₉H₁₉N₅Na₂O₈S₂;available, for example, from Sigma-Aldrich). Direct Red 81 and manyother tags or labels may be used with many different bacteriophages anddo not interfere with the activity of the bacteriophage, was used instudies hereof. In general, virtually any tag or label (for example, atag or label detectible via a photoacoustic signal) can be used inconnection with any bacteriophage. There is also an almost unlimitedvariety of tags that may be used. Because of the chemistry of suchlabels, labeling the bacteriophage may be as simple as mixing thebacteriophage with the label.

Many techniques known to those skilled in the art are suitable forattachment of labels or tags to bacteriophages. Fluoresceinisothiocyanate forms covalent adducts with amino groups. With proteins,the epsilon-amino groups of lysine residues may provide the aminogroups. The capsid proteins of the bacteriophages provide lysineresidues for reaction. FIG. 1 illustrates an example of a reactionscheme for the reaction of an amino group with an isothiocyanate. FIG. 1also illustrated the chemical structure of fluorescein isothiocyanate.In the scheme of FIG. 1, R′ is the fluorescein moiety and R″ is thetarget protein with NH₂ being the epsilon amine of a lysine residue ofthat protein. Further, many dye compounds suitable for use as labelsherein, contain sulfonic acid (SO₃) groups. The pKa of these sulfonicacid groups is sufficiently low that they afford extremely tight bindingwith basic groups provided by lysine and arginine residues in proteins,especially at the relatively neutral pH and ionic strengths used in thetests (assays) hereof.

In a number of embodiments, a preparation of a single bacteriophage witha known host range was titered to determine specifically and accuratelythe number of infectious particles/ml. The photoacoustic label was boundto phage by incubating phage with an excess of the label. Labeledbacteriophage were then pelleted using a desktop centrifuge andre-suspended in fresh buffer to remove any unbound label. Labeled phagewere titered to determine the number of infectious particles/ml. Thenumber of infectious particles/ml pre- and post-labeling wasindistinguishable, demonstrating that labeling of phage particles didnot decrease or change their rate of infectivity. In severalrepresentative embodiments hereof, labeled Det7 bacteriophage was boundto LT2 Salmonella, labeled HK97 was bound to LE392 E. coli, D29bacteriophage was used in connection with k12 E. coli as a negativecontrol (D29 is a mycobacterium bacteriophage and does not attach to E.coli), and labeled T4 bacteriophage was bound to E. coli B and K12 E.coli and Serratia.

As described above, in a number of studies, the labeled phage wasdetected using photoacoustic labels or tags. In that regard, labeledbacteriophages with a known titer from the previous studies werecollected in fresh buffer. Phages were pelleted using a desktopcentrifuge and re-suspended in fresh buffer with no photoabsorbance.Phages were re-suspended to give a known number of labeled particles/ml.The phage buffer mixture was then titered through the photoacoustic cellto determine the number of labeled phages needed for detection. Labeledphages were detected, demonstrating that signal from labeled unboundphages was detectable by the photoacoustic cell.

The labeled phage was then tested with target bacteria. In a number ofstudies, host bacteria of labeled phages were titered to determine thecolony forming units/ml. Labeled phages from previous studies of a knowntiter were added in a 10 to 1 ratio of phage to target bacteria. Thephage/bacteria mixture was incubated together for a set period of timeto allow the phages to bind to the bacteria. The phage/bacteria mixturewas then pelleted using a desktop centrifuge and re-suspended in anequal volume of fresh buffer to remove any unbound labeled phages.Bacteria with bound labeled phages were then run through thephotoacoustic cell and signal detected. Unbound bacteria alone were runas a negative control.

FIG. 2 illustrates a representative embodiment of a photoacousticflowmetry system. In one study, a rapid bacterial typing assay hereofused photoacoustic flow cytometry- or laser induced ultrasound, in which532 nm laser light generated ultrasonic waves in phage containing aphotostable chromophore. Bacteriophages were labeled using aphotoacoustic labile tag and then added to bacterial cultures of targetand non-target bacteria as described above. Once again, excess andunbound phage was removed after bacterial absorption usingcentrifugation. Labeled phage/bacterial mixture was then processedthrough a microfluidic system in which 5 ns pulsed laser light generatedhigh frequency ultrasonic waves in the targeted bacterial cells as aresult of thermoelastic expansion in the dye particles. Thephotoacoustic flowmeter contained a focused ultrasound transducer thatdetected the ultrasonic waves generated in target bacteria. This signal(see, for example, FIG. 3) was processed and stored in a computer usingan automated software interface. Bacterial cells providing a positivesignal were charted against the total number of cells processed toobtain an absolute number of each target bacteria cell in the culture.Using E. coli, for example, a signal to noise ratio in excess of 5:1 wasachieved. The studies hereof demonstrated that bacteriophages—coupledwith a detection scheme such as photoacoustics may be effectively usedto quantify specific bacteria in a mixed culture.

FIG. 4 illustrates progression of thermoelastic expansion caused bylaser pulse inside each droplet during photoacoustic detection. FIG. 5illustrates a view of photoacoustic cell using two phase flow. Thesample flows through the photoacoustic cell one droplet at a time. Eachdroplet is impinged with light from the laser, and a signal is detectedby the acoustic sensor (not shown, but may, for example, be positioneddirectly under the photoacoustic cell).

FIGS. 6A and 6B illustrate two graphs of photoacoustic waveforms fromirradiating free-floating or unbound phage. The waveform of FIG. 6A,results from laser irradiation of bacteriophage at about 2.12millijoules (mJ) per pulse. The waveform of FIG. 6B results fromirradiation of bacteriophage at 3.96 millijoules (mJ) per pulse. Theseresults indicated that in embodiments of detection algorithms hereof,use of sufficiently low energy (for example, approximately 2 mJ)irradiation results in waveforms that are below a predetermineddetection threshold. A suitable level of energy for irradiation isreadily determined by one skilled in the art. Bacteriophage that arebound to bacteria will be more concentrated and will give largeramplitude signals. Beneficially, any free-floating phage will not bedetected in a detection protocol with sufficiently low energy.

A demanding challenge for clinicians is to correctly identify patientswho are most likely to benefit from empiric treatment withbroad-spectrum antibiotics with activity against multi-drug resistantpathogens. These antibiotics are more expensive and carry a greatertoxicity than standard regimens. In addition, the use of broad-spectrumantibiotics for patients who could be treated with narrower-spectrumdrugs results in emergence of drug resistance in the community andfurther complicates management. The ability to exclude organisms with arisk of drug resistance would allow the use of cheaper, saferantibiotics for many patients now treated with empiric broad-spectrumtherapy.

Two scenarios confound diagnosis of bacterial infections. First, somebacteria are hard or even impossible to grow in culture. For example,common pathogens in pneumonia such as Mycoplasma pneumoniae do not growin culture. Second, even organisms that can routinely be cultured willnot grow in the presence of antibiotics (that may have been given to thepatient empirically). Resins that can remove antibiotics from the sampleare sometimes effective but often all therapy must be stopped for aperiod of time and then the patient's blood or other fluids can becultured. This delays management and may put the patient at risk.

When non-sterile fluids are cultured (e.g. sputum) there is often a needto estimate the number of bacteria present in order to distinguish florafrom pathogen. The usual approach is to use serial dilutions to “plateout” organisms and count the colonies. This a slow process that canrequire days to obtain a result. Phage-based detection as describedherein has the ability to estimate the number of organisms in a samplein a single step, taking, for example, hours rather than days.

In a representative clinical example, a patient presents with cough,high fever and a chest radiograph showing a lobar infiltrate. Thestandard approach to management would be to use broad-spectrumantibiotics to cover a variety of pathogens and send samples of bloodand sputum for culture. The disadvantages of this approach include: (i.)multiple antibiotics or “super antibiotics” are needed introducinghigher cost and greater risk for side effects; (ii.) ability to “cover”only about 85-90% and thus treatment may be ineffective in 10-15% ofcases leading to delay in adequate antibiotics and increased risk ofdeath; and (iii.) initial cultures are only positive in about 50% ofcases (or less) and subsequent cultures may fail because antibiotics areon board and will be in the sample, leading to a diagnostic change andincreased risk for adverse outcomes.

By contrast, the approach of a representative embodiment of the presentmethodology is shown in the Table 1 below. In Table 1, NA is notapplicable and BSA is a broad-spectrum antibiotics (for example,piperacillin tazobactam). In the example of Table 1, the differentlabeled phages (that is, including labels with different/separatelydetectible or measureable detection characteristics/wavelengths) couldbe applied concurrently to the sample.

TABLE 1 Bacteria Phage Label Wavelength Comment Treatment PneumococcusSV1 Trypan Blue 430 Difficult to grow in Ampicillin cultureStaphylococcus NA81 Direct Red 532 May be methicillin Vancomycin 81resistant Haemophilus Inf. HP2 Methylene 605 May be resistance toCeftriaxone Blue ampicillin Mycoplasma MFV1 Indocyanine 670 Does notgrow in Erythromycin Green culture Legionella ΦLP6 India Ink 800 Doesnot grow in Erythromycin culture Other bacteria NA Null Most otherbacteria BSA will grow None NA Null Viruses and fungi are Noantibiotics* rare causes

In the example of Table 1, a sample showing a photoacoustic signal froman excitation laser wavelength of 430 nm would be interpreted aspositive for pneumococcal pneumonia (the most common pathogen) and wouldallow the use of a low cost, low-toxicity single drug regimen(ampicillin). Conversely a photoacoustic signal from an excitation laserwavelength of 532 nm would lead to a diagnosis of staphylococcus. Thisorganism is commonly resistant to ampicillin (and other penicillin-likedrugs) and so vancomycin would be used. The treatment of viral or fungalpneumonia with antibiotics can actually cause harm. Further workup forrare causes including tuberculosis would be facilitated by earlyrecognition that a “rare cause” is present. A result of “null” wouldlead clinicians to either suspect unusual bacteria or (more commonly)viral infection. Importantly, a null result from the test hereof and “nogrowth” result from culture (available in 24 hours) would lead to ahighly likely viral diagnosis, and antibiotics could be withheld. Thebacteriophages listed in Table 1 are only representative phages for eachbacteria. As known to those skilled in the art, there are numerous otherbacteriophages that could be used in connection with each of the listedbacteria.

Experimental

Representative protocol for bacteriophage binding. In a representativeexample, the bacteriophage was HK97 as 10¹⁰ plaque forming units per ml(pfu/ml). The bacteria was Ymel E. coli at 10⁹ colony forming units perml (cfu/ml). The culture could be stored at 4° C. for approximately 1week. For incubation, 10-100 bacteriophage were added per bacterialcell. The mixture was incubated for 5-10 minutes at 37° C. with shaking.Incubation with water at 37° C. with gentile agitation is alsoacceptable. The cell/bacteriophage mixture was placed in an eppindorftube and spun 10,000×g for 1-2 minutes. The supernatant (containingunbound phage) was poured off. The pellet was resuspended in equalvolume of chilled phosphate buffer solution (PBS), luria broth (LB), orsterile water. Infected cells will have 20-60 minutes before lysisstarts to occur.

Representative protocol for labeling bacteriophage and unboundbacteriophage studies. 100 μl of purified bacteriophage (10¹¹ pfu/mlfinal concentration) was added to 900 μl of direct red solution at aconcentration of 100 μg/ml. The bacteriophage was incubated at roomtemperature for 30 minutes after vortexing. The bacteriophage was thenpelleted using a refrigerated centrifuge (for example, for 3 hours at14,800×g at 4° C. The supernatant was removed and the pellet was washedwith PBS at a pH OF 7.2 (10¹¹ pfu/ml).

In the photoacoustic studies of the bacteriophage at 2.12 mJ (see FIG.6A), a 1 ml sample was run through the photoacoustic system with laserenergy at 2.12 mJ. In 23 detections, 11 were greater than the thresholdlevel. Constant low-level detection was just below the threshold. Thesignal from low-level detection disappeared when water was runsubsequently through the system. The sample was collected and used forthe next experiment. The recovered sample was tittered and found tostill be 10¹¹ pfu/ml. The few detections that were above threshold levelwere likely a result of clumped bacteriophage.

In the photoacoustic studies of the bacteriophage at 3.96 mJ, the laserenergy was increased to 3.96 mJ (see FIG. 6B). Water was run through thesystem to test background. In running the bacteriophage sample throughthe system, constant detection, well above the threshold, was achieved.Water was run through the system directly after detection ofbacteriophage and the signal disappeared completely.

These results indicated that aggregated bacteriophage can be detected ata lower energy level (for example, approximately 2 mJ). Thebacteriophage will be aggregated using target bacteria. At such lowerenergy levels, however, unbound phage will not be detected.

The foregoing description and accompanying drawings set forth a numberof representative embodiments at the present time. Variousmodifications, additions and alternative designs will, of course, becomeapparent to those skilled in the art in light of the foregoing teachingswithout departing from the scope hereof, which is indicated by thefollowing claims rather than by the foregoing description. All changesand variations that fall within the meaning and range of equivalency ofthe claims are to be embraced within their scope.

1. A method of determining if a sample taken from a patient includes aspecies, strain or type of bacteria, comprising: mixing a labeledbacteriophage including a label that is detectible via a detectionsystem with the sample, the labeled bacteriophage being active toselectively bind with the species, strain or type of bacteria and usingthe detection system to determine the presence of the labeledbacteriophage bound to the species, strain or type of bacteria.
 2. Themethod of claim 1 further comprising removing unbound labeledbacteriophage after mixing the labeled bacteriophage with the sample. 3.The method of claim 1 wherein the detection system comprises aphotoacoustic cell.
 4. The method of claim 1 wherein the detectionsystem comprises a flowmetry system.
 5. The method of claim 1 whereinthe species, strain or type of bacteria is quantified upon detection. 6.The method of claim 3 wherein the level of energy used in thephotoacoustic cell is sufficiently low to reduce detection of unboundlabeled bacteriophage.
 7. The method of claim 1 wherein a plurality oflabeled bacteriophages are mixed with the sample, each of the pluralityof bacteriophages selectively binding with a different species, strainor type of bacteria, each of the plurality of bacteriophages including adifferent label that is separately detectable via the detection systemand using the detection system to determine the presence of the labeledbacteriophage bound to at least one of the different species, strains ortypes of bacteria.
 8. A bacteriophage comprising a label attachedthereto, wherein the label is selected to be detectible via a detectionsystem.
 9. The bacteriophage of claim 8 wherein the label is detectibleusing a photoacoustic cell.
 10. The bacteriophage of claim 8 wherein thelabel is detectible using a flowmetry system.
 11. A system fordetermining if a sample includes at least one species, strain or type ofbacteria, comprising: a plurality of bacteriophages, each of theplurality of bacteriophages selectively binding with a differentspecies, strain or type of bacteria, each of the plurality ofbacteriophages including a different label that is separately detectablevia a detection system.
 12. The system of claim 11 wherein each of thedifferent labels of the plurality of bacteriophages is detectible usinga photoacoustic cell.
 13. The system of claim 11 wherein each of thedifferent labels of the plurality of bacteriophages is detectible usinga flowmetry system.
 14. A system for identifying at least one species,strain or type of bacteria in a sample, comprising: at least one alabeled bacteriophage which binds selectively to the species, strain ortype of bacteria, the at least one labeled bacteriophage comprising alabel attached thereto, the system further comprising a detection systemadapted to detect the labeled bacteriophage bound to the species ofbacteria.
 15. The system of claim 14 wherein the detection systemcomprises a photoacoustic cell.
 16. The system of claim 14 wherein thedetection system comprises a flowmetry system.
 17. The system of claim14 wherein the species, strain or type of bacteria is quantified. 18.The method of claim 7 further comprising removing unbound labeledbacteriophage after mixing the labeled bacteriophage with the sample.19. The method of claim 7 wherein the detection system comprises aphotoacoustic cell and the level of energy used in the photoacousticcell is sufficiently low to reduce detection of unbound labeledbacteriophage.
 20. The method of claim 7 wherein the detection systemcomprises a photoacoustic flow cytometry system.